Method for the preservation of viruses and mycoplasma

ABSTRACT

A biologically-active material comprising a live virus or mycoplasma is preserved by a method of desiccation, without lyophilisation, in a matrix of glassy trehalose having a residual moisture content of not greater than 2%. The method comprises two vacuum drying stages. In a cycle time much shorter than a typical freeze drying process a virus or mycoplasma can be preserved to provide a material that can be rehydrated to give a vaccine having potency.

The present invention relates to the preservation of viruses andmycoplasma. In particular, it relates to an ultra-rapid method by whichsuch materials can be preserved using the disaccharide, trehalose. Bythis method, a long term preservation of viruses and/or mycoplasma canbe achieved and, especially, living attenuated vaccines can be prepared.

The preservation of biodegradable materials by dehydration andosmoconcentration is a familiar and ancient technology. When the task ofpreserving sensitive biomolecules became necessary, simple drying bydehydration failed, as structural water was removed, causing subsequentdenaturation and loss of vital activity. Cryopreservation in liquidnitrogen and lyophilisation have become the accepted methods for thelong term preservation of sensitive biomolecules, the latter methodbeing used extensively for the preservation of live attenuated vaccines.

Improved thermotolerance of freeze dried Rinderpest vaccine has beenachieved by extending the secondary drying cycle, in order to reduceresidual moisture (RM) levels to around 1%-2%. This entails long andhigh energy consuming operational cycles of up to 72 hours as describedby Mariner, J. C. et al., Vet. Microbiol., 1990, 21, 195-209. Vaccinesproduced by this method are known and are distinguished from thestandard vaccine by the name “THERMOVAX”.

As mentioned above, these currently used processes are time consumingand involve high energy input. Furthermore, lyophilisation confers onlya modest level of thermotolerance in the final product and refrigerationis still required to reduce deterioration during storage. This is aparticular problem for live vaccines to be used in tropical climatessince these lose potency with the unfortunate result that vaccinationprograms carried out in the field in tropical countries, wheremonitoring the “cold chain” is difficult, ultimately lead to vaccinationof patients with substandard or, in some cases, useless vaccine.

During evolutionary natural selection, certain species of plants andanimals acquired the remarkable and elegant ability to tolerate extremedehydration, remaining dormant in hostile environments for very longperiods of time and yet able to assume complete vital activity onrehydration. Examples include the resurrection plant Selaginellalepidophya, the brine shrimp Artemia salina (Clegg, J.,J.Comp.Biochem.Physiol, 1967, 20, 801-809), the yeast Saccharomvcescerevisiae (Coutinho, E., Journal of Biotechnology, 1988, 7, 23-32) andthe tardigrade Macrobiotus hufelandi (Kinchin, I. M., Biologist, 1995,42, 4). Such organisms are termed cryptobiotic and the process by whichthey survive is known as anhydrobiosis. All species of animals andplants which display this ability contain the disaccharide trehalose(α-D-glucopyranosyl-α-D-glucopyranoside). Its presence generally in theorder of 0.2 g/g dry cell weight in most cryptobionts enables them toresist extreme dehydration, high temperatures, X-rays and also in somespecies of tardigrades, pressures as high as 600 Mpa.

Colaco et al., Biotechnology, 1992, 10, 1007-1011, describe the benefitof rapid drying of biological materials using trehalose. This methodmostly refers to the drying of restriction enzymes and immunoglobulinsonto preformed solid matrices, such as cellulose fibres or onto thesurfaces of plastic plates for diagnostic purposes such as ELISA orsimilar diagnostic applications in the laboratory. Problems arise,however, with these techniques when scaling up to industrialapplications, such as large scale commercial vaccine production wheremuch larger unit numbers and volumes have to be handled using mandatoryaseptic techniques in partially sealed vials. To meet the operationalrequirements of large scale vaccine production, where unit volumes from1.0 ml upwards and production batches of 20 litres are typical, adifferent strategy is required to remove that volume of water in aneconomically acceptable time. Drying at atmospheric pressure even at thehighest physiologically tolerated temperatures would require anunacceptably long time to remove water quickly enough from partiallystoppered vaccine vials and would inevitably result in denaturation andloss of potency.

The present invention is concerned with a method of preservation ofviruses or mycoplasma using trehalose under conditions which cause waterto be removed while, at the same time, allew the biological integrity ofthe material to be maintained.

Accordingly, the present invention provides a method of preserving abiologically-active material comprising a live virus or mycoplasma whichmethod comprises the steps:

(i) mixing an aqueous suspension of the biologically-active materialwith a sterile aqueous solution of trehalose to give a trehaloseconcentration in the mixture in the range of from 0.2 to 10% w/v;

(ii) subjecting the mixture to primary drying, for 30 to 60 minutes, ata pressure of less than atmospheric and at a temperature initially nogreater than 37° C., and which is controlled not to fall to 0° C. orbelow and which finally is no greater than 40° C. to form a glassyporous matrix comprising glassy trehalose having a residual moisturecontent of not greater than 10% and containing, within the matrix,desiccated biologically-active material; and

(iii) subjecting the glassy porous matrix of step (ii) to secondarydrying for 10 to 30 hours at a pressure not greater than 0.1 mbar and ata temperature which finally is in the range of from 40 to 45° C. to forma trehalose matrix having a residual moisture content of not greaterthan 2% containing, within the matrix, desiccated biologically-activematerial.

By using the method of the invention it is possible to produce a livevaccine with, compared to prior art methods, enhanced biologicalcharacteristics and distinct commercial advantages. Vaccines preparedusing the method of the invention are dried much more quickly than thoseusing conventional freeze drying procedures. For instance, the method ofthe invention can be used to dry trehalose/biologically-active materialmixtures to a moisture content of about 10% in less than one hour.Further dehydration to a residual moisture content of about 1-2% can beachieved in less than 30 hours, for instance about 20 hours, compared toa period of 50 hours by conventional freeze drying procedures.Furthermore, damage caused by solute concentration is minimisedaccording to the present invention and particularly damaging icecrystallisation is avoided. The thermostability of thebiologically-active material preserved in the trehalose glassy matrix isgreater than that of materials preserved by prior art methods and, thus,the necessity of the “cold chain”, which is a serious constraint withconventional freeze-dried vaccine, is minimised. The product of thepresent invention can be exposed to high ambient temperatures, e.g., upto about 45° C., for prolonged periods without any substantial loss ofbiological activity. In addition to these and other advantages of thepresent invention the product of the method exhibits instantaneous“flash solubility” upon rehydration.

The method of the present invention is suitable for achieving the longterm preservation of viruses and mycoplasma. In particular, it can beused to preserve highly labile live attenuated viral components andmycoplasma components that can be rehydrated to form vaccines. Examplesof such biologically-active materials that can be preserved according tothe method of the invention include:

Family: Paramyxoviridiae

Subfamily: Paramyxovirinae

Genera: Parainfluenza virus group Measles, Rinderpest, canine

distemper, Peste des Petits Ruminants (PPR)

Paramyxovirus: mumps virus (Mumps)

Genus: Rubivirus, Rubella (German Measles)

Genus: Flavivirus, Yellow fever virus (Yellow Fever)

Genus: Rhabdoviruses, Lyssaviridiae (Rabies virus)

Picoma viruses (Polio virus)

Newcastle Disease virus

Mycoplasma: Mycoplasma mycoides (Contagious Bovine Pleuropneumonia)

Brucella abortus: Strain 19 vaccine

Chlamydia: Chlamydia psittaci (Enzootic abortion)

Coccidia: Toxoplasma gondii (Toxoplasmosis)

According to the method of the present invention, thebiologically-active material to be preserved is prepared as a suspensionin an appropriate aqueous medium. In the case of a virus, it may be thatthis will need to be cultured, for instance in vero cells, in anappropriate culture medium, and then harvested prior to suspension inorder to provide a useful concentration of material. Typically, theaqueous suspension of biologically-active material will be pH adjusted,for example by the addition of an alkali, to a pH in the range of from7.0 to 7.8 especially about 7.4.

Trehalose is one of the most stable and chemically non-reactivedisaccharides. It has an extremely low bond energy of less than1Kcal/Mol making the dimer structure very stable. It does not undergocaramelisation unless heated severely, nor does it cause the Maillardreaction with proteins or peptides. The natural di-hydrate structurecontaining two water molecules enables unusual flexibility around thedisaccharide bond which possibly permits a closer association withtertiary structured biomolecules. It is not hygroscopic yet exhibits“flash solubility” on hydration, a property particularly useful fordried vaccines.

The aqueous suspension of the biologically-active material is mixed witha sterile aqueous solution of trehalose and the mixture will be preparedsuch that it will have a trehalose concentration of from 0.20% to 10%w/v, preferably 2 to 10% and more preferably from 2.5 to 8% w/v. Withinthe range of trehalose concentrations, the actual concentration usedwill, in general, depend on the unit size of the biologically-activematerial. Less trehalose is required for small virus particles than forlarge cells.

The sterile aqueous trehalose/biologically-active material mixture issubjected to a vacuum drying procedure involving a primary drying stepfollowed by a secondary drying step. Preferably, a conventional freezedrying apparatus (e.g., such as manufactured by EDWARDS, CHRIST,USIFROID or SAVANT) is used for the drying procedure in order to providea controlled environment for the critical stages in the method. It isemphasised, however, that if such an apparatus is used freeze dryingconditions are not employed and the material is not subjected to freezedrying. The drying procedure comprises two drying stages, a primarydrying stage in which the residual moisture content of the material isreduced to a value of not greater than 10% and a secondary drying stagein which the residual moisture content of the material is reducedfurther to a value not exceeding 2%.

In the primary drying stage the initial temperature of the dryingapparatus will be such as to ensure that the temperature of thetrehalose mixture will not be greater than 37° C. and will preferably be37° C. in order to prevent any loss of biological activity at this stagein the method. As water is evaporated off the mixture, the temperatureof the mixture falls. The desiccation is, however, carried out to ensurethat no freezing or sublimation from ice occurs as is normallyexperienced in conventional freeze drying procedures, i.e., thetemperature of the product during this stage of the drying process iscontrolled not to fall to 0° C. or below and is typically controlled notto fall below 4° C. The pressure is reduced below atmospheric pressure,preferably to a value of from 800 to 300 mbar.

The primary drying stage is continued for a period of time, between 30and 60 minutes, during which time the temperature of the trehalosemixture initially falls as water is evaporated off and then rises. Whena temperature (under the reduced pressure employed) of about 25° C. isreached during this procedure the trehalose forms a glassy matrixcomprising glassy trehalose and containing the biologically-activematerial which is in a desiccated or partially desiccated state. Theprimary drying stage is completed when the moisture content of theglassy matrix has reached 10% or below and it is essential, at thiscritical stage in the procedure, that the temperature is not allowed toexceed 40° C. If the temperature at the end of the primary drying stagedoes exceed 40° C. the biologically-active material will sustain damage.

The product obtained from the primary drying stage is then subjected toa secondary drying stage, preferably without removal from the dryingapparatus used for the primary drying stage. In the secondary dryingstage the glassy matrix obtained from the primary drying stage isfurther dehydrated under a reduced pressure, not exceeding 0.1 mbar.Currently-available specialised apparatus has the ability to operate atextremely low pressures, for instance below 0.001 mbar. However, verygood results have been obtained according to the present invention usingreduced pressures for the secondary drying stage in the range of from0.001 to 0.1 mbar, typically from 0.01 to 0.1 mbar. The secondary dryingstage is continued for a total period of time in the range of from 10 to30 hours, preferably 20 to 30 hours.

As mentioned above, the temperature of the glassy matrix at the end ofthe primary drying stage does not exceed 40° C. During the secondarydrying stage the temperature of the matrix may rise slightly to a finaltemperature, at the end of the drying procedure, of from 40° C. to 45°C. The trehalose matrix, at the end of the secondary drying stage willhave a residual moisture content of 2% or less, preferably 1% or less inorder to ensure a very high degree of thermostability in the product. Atsuch residual moisture contents the final temperature in the dryingprocess should not be higher than 45° C. since the desiccatedbiologically-active material in the matrix may undergo some degree ofdestruction above 45° C. Preferably, the temperature of the product atthe end of the secondary drying stage will not exceed 44° C.

It has been found, through experimentation that there is some benefit incontrolling the temperature of the matrix, during the secondary dryingstage, such that it is maintained at about 37° C. for a period of from15 to 17 hours and then raising this gradually (for instance by 0.5 to1° C. per hour), over the remaining secondary drying time to a finaltemperature within the range of 40° C. to 45° C.

The glassy trehalose matrix produced according to the method can berehydrated very quickly in an appropriate aqueous medium, typicallysterile distilled water, to produce a vaccine for use in a very shortperiod of time.

The preparation of the vaccine and the operating procedure using afreeze dryer for the desiccation of viruses, such as Rinderpest andPeste des Petits Ruminants viruses, and mycoplasma without alyophilisation step involving sublimination from ice, exploits theunique property of the disaccharide trehalose to protect tertiarymacromolecules during desiccation.

Compared to conventional freeze-drying procedures the method of theinvention offers the following benefits:

It provides a high level of virus protection, employing a relativelyshort, simple procedure, e.g., a 25 hour production cycle, thus reducingproduction cycle time and energy costs.

Basic drying equipment is all that is required, although sophisticatedmicroprocessor controlled freeze dryers can also be used, but are notstrictly essential.

The method is tolerant of power interruption, unlike lyophilisationwhere even a short power failure can cause product meting, leading tounacceptable loss of virus.

Oral vaccination with some attenuated strains of virus has in the pastbeen difficult to achieve because of the loss of epitheliotropism. Bothoral and intranasal vaccination would be useful and appropriate for manyapplications because they mimic the natural route of droplet infection,generating a cascade of protective mucosal immunity, with IgA andhumoral IgG2a T helper-cell type 1 response. It would be easy toadminister such routes of vaccination and these would be applicable inthe event that a suitable vaccine is prepared. A vaccination procedurewith live attenuated strains mimicking the natural route of infectioninduces a more comprehensive sero mucous and cell mediated immunity.According to a further aspect the present invention provides a method ofmaking a vaccine for oral or intranasal use which comprises preparing aglassy matrix of trehalose containing desiccated virus according to theabove described method, combined with a suitable positively-charged,biocompatible, water-soluble adjuvant, and rehydrating the glassy matrixwith an appropriate aqueous composition. According to a preferredembodiment of this aspect of the invention the vaccine for oral orintranasal vaccination is an MMR vaccine. Since the current paediatricMMR vaccine is prepared by conventional freeze drying technology and isinjected into the patient subcutaneously, an oral or intranasal vaccinewould give great benefits.

EXAMPLES Example 1

A paramyxovirus Rinderpest RBOK attenuated vaccine strain was grown insecondary calf kidney cells for 10 days at 37° C. The virus suspensionwas harvested and clarified by centrifugation at 1000 rpm in arefrigerated centrifuge.

A sterile excipient containing 10% w/v trehalose and 5% lactalbuminhydrolysate was added at a ratio of 1:1 with the clarified virus fluidat 4° C.

The excipient/virus mixture 1.0 ml of this was aliquoted into sterile 10ml neutral glass vials which were then partially stoppered with steriledry butyl rubber stoppers.

The vials were placed on the shelf of the freeze drying chamber and thetemperature of the product was raised to 37° C.

The condenser was started and the condenser temperature allowed tostabilise at −60° C.

The vacuum pump was started and the chamber containing the product wasevacuated to a pressure of 800 mbar and the product gently degassed for30 minutes to avoid sputtering whilst carefully checking the producttemperature to avoid evaporative freezing. By maintaining a pressuregradient between the drying chamber and the refrigerated condensor 75%of the water vapour was driven over to the condenser in the first 30minutes.

The pressure was then lowered to 500 mbar and a structured metastableglassy matrix was formed. The pressure was then further reduced to 0.10mbar and maintained for 30 minutes.

The vials were stoppered and sealed under a final vacuum of 0.01 mbarand capped with aluminium closures.

Virus titre before drying: 10⁴TCID/50/ml

Virus titre after drying: 10⁴TCID/50/ml

The product matrix was flash soluble in distilled water. This exampleshows that the potency of the virus is substantially unaffected by thetreatment employed in the primary drying stage of the method of theinvention. It further illustrates the possibility of dehydrating thevirus, to prepare viral vaccines, on an industrial scale.

Example 2 Materials and Methods

Preparation of the RP and PPR Vaccine Cultures

Peste des petits ruminants (PPRV 751/1) and Rinderpest (RBOK) strainswere grown initially using vero cells in Glasgow modification Eaglesmedium (GMEM) supplemented with 10% tryptose phosphate broth (TPB,Difco) and 10% foetal calf serum (FCS) as follows:

Vero cells were seeded into 5×150cm² plastic flasks at a cellconcentration of 287,000 cells/ml, 60 ml per flask. Two flasks wereinoculated with 0.5 ml PPR virus suspension at a multiplicity ofinfection of 0.03 virus particles/cell. Two flasks were similarlyinoculated with RP virus. One flask remained uninoculated as a control.

The flasks were incubated at 37° C. in 5% CO₂ and cells examined dailyfor development of cytopathic effects (cpe). On day 4 the GMEM mediumwas replaced with Hanks lactalbumin yeast extract (Hanks LYE),containing 2% FCS and 0.1% trehalose dihydrate. On day 6 the cpe wasapproximately 80% and the virus harvests were pooled, frozen and storedat −20° C. The control flask remained in the incubator for 10 days, andproved to be free from contamination or cell degradation, with noobvious sign of adventitious agents.

Dehydration Procedure

The dehydration procedure used can be considered to consist of two maincomponents similar to lyophilisation: primary drying and secondarydrying. The fundamental difference from lyophilisation is that theproduct is not frozen and drying is by simple dehydration, notsublimation from ice.

The pooled virus suspensions were thawed and diluted 1:1 with a sterile5% w/v aqueous solution of trehalose dihydrate, thus giving a finalconcentration of 2.5% w/v trehalose in the mixture. One ml volumes weredistributed into each of 5 ml vaccine vials and partially sealed withdry vented butyl rubber inserts. This operation was carried out at roomtemperature, in a laminar air flow biohazard cabinet and observingstrict aseptic precautions.

Primary Drying

The dehydration process was carried out using an Edwards Supermodulyofreeze dryer with precise control over chamber pressure, condenserpressure, shelf and product temperatures. The freeze dryer was preparedin advance before loading the shelf chamber with the vials containingthe product. The shelf temperature was raised to 40° C. and thecondenser temperature was allowed to reach the operational limit of −40°C. Vials were then placed on the shelves and the contents allowed toreach 35° C. The chamber door was closed with the macro and micro airadmittance valves fully opened and the vacuum pump switched on with fullgas ballast. The pressure in the chamber was adjusted to 800 mbar bycarefully closing the macro air admittance valve. The pressure in thecondenser was maintained at 500 mbar in order to produce a pressuregradient between the chamber and condenser and this provided the drivingforce to induce water vapour to flow from the product surface to thecondenser. Partial closure of the vials with the stoppers also had thebeneficial effect of throttling the aperture thus increasing thepressure still further at the product surface. It was noticed that thepartially closed vials dried quicker than the fully open ones containingthe temperature recording thermocouples.

The change in the temperature of the product with time and the change inthe chamber pressure with time during the primary drying stage are shownin the attached Figure. As can be seen in the Figure, evaporationstarted immediately as indicated by the fall in product temperature. Theproduct temperature was controlled primarily by carefully closing themacro air admittance valve during the first 15 minutes, and thereafterby manipulation of the micro air admittance valve, making sure not toallow the product to freeze. Maintaining a temperature around 1-2° C.caused by evaporative cooling, increased the evaporation rate so that90% of the water had evaporated within one hour and the producttemperature began to rise to match the shelf temperature. As dehydrationproceeded a critical point was reached after 40 minutes when there was asudden rapid rise to 25° C. followed within seconds by a sudden fall inproduct temperature to 15° C. This was accompanied by a dramaticbubbling of the product.

Secondary Drying

A further batch (batch 2) of the live attenuated Rinderpest strain (RP)was prepared and subjected to primary drying for 1 hour as describedabove. This was then subjected to a period of secondary drying where thetemperature was raised over a period of a further 17 hours to a finalproduct temperature of 42.4° C. and a pressure of 0.06 mbar, with gasballast fully closed. This had the effect of reducing the residualmoisture content of the material to approximately 0.72%.

A further batch (batch 2) of the live attenuated Peste des petitsruminants strain (PPR) was prepared and subjected to primary drying for1 hour as described above. This was then subjected to a period ofsecondary drying. The secondary drying was stopped after 2 hours at aproduct temperature of 42.8° C. and a chamber pressure of 0.06 mbar. Theproduct, after this short secondary drying procedure, had a residualmoisture content of 5.36%.

The Test for Thermostability

Samples from batch 2 of each of PPR and RP prepared as described abovewere stored at 4° C., 25° C., 37° C. and 45° C. Three vials from eachstorage temperature were taken for virus titration on days 0, 3, 7, 10and 14-post incubation. The geometric mean of the three vials wasconsidered as the residual virus titre for each batch type at eachtemperature for the specified period of incubation (Tables 3 and 4).

Virus Titrations

These were carried out to determine the efficiency of the ultra rapidone hour dehydration as assessed by the degree of protection induced bythe trehalose glassy state. The virus titrations were performed asdescribed in the standard operating procedures for Rinderpest vaccinedescribed by Mark, M. Rweyemamu et al., in FAO Animal Production andHealth Paper, 1994, No. 118. (NB FAO is the Food and AgricultureOrganisation of the United Nations). Note: The main body for theregulation and co-ordination of animal vaccine quality is The OfficeInternational Epizooties (OIE). The OIE standard for potency for RP is10^(2.5) TCID₅₀ s/dose and for PPR is 10³ TCID₅₀ s/dose (where TCID isthe tissue culture infective dose). In the Tables 1, 2, 3 and 4 belowthe virus titres are expressed as X log₁₀ TCID₅₀/ml where “X” is thefigure shown under Test 1, 2 and 3 in each of the Tables.

Results and Discussion

The results obtained from paired samples of RP and PPR vaccine dried forone hour, using the method described above, in comparison with a controlsample of lyophilised vaccine and also with the parent untreated viruspool containing 2.5% trehalose were as depicted in tables 1 and 2. Theexcipient containing 2.5% trehalose gave good protection of RP virusfollowing the rapid dehydration at 37° C. under conditions of reducedpressure at 800 mbar with the loss following drying of 0.45 log₁₀TCID₅₀/ml.

TABLE 1 Rinderpest virus titration results after primary dryingSubstance tested Test 1 Test 2 Test 3 Virus pool + 2.5% Trehalose 5.75.9 5.8 Virus pool + 2.5% Trehalose 5.0 5.7 5.35 dried at 37° C. for 1hour Lyophilised Reference Vaccine 4.8 4.8 4.8

The protection of the PPR virus in the same excipient was excellent withonly the loss of 0.15log₁₀ CID₅₀/ml following drying.

TABLE 2 Peste des petits ruminants virus titration Substance tested Test1 Test 2 Test 3 Virus pool + 2.5% Trehalose 5.0 4.9 4.95 Virus pool +2.5% Trehalose 4.9 4.7 4.8 dried at 37° C. for 1 hour LyophilisedReference Vaccine 4.9 4.9 4.9

The incorporation of a secondary phase in the dehydration processclearly has a marked effect on the thermotolerance of the product. Thisis demostrated by the fact that the RP batch 2 which was subjected to 17hours of secondary drying lost only log 1.9 TCID/50/ml after two weeksat 45° C.

TABLE 3 Thermostability test of Rinderpest Batch 2 Virus titre afterstorage at various Temperatures Day of Incubation 4° C. 25° C. 37° C.45° C. 0 4.97 4.97 4.97 4.97 3 4.83 4.70 4.10 3.80 7 4.83 4.63 4.17 3.3710 4.80 4.57 4.10 3.30 14 4.87 4.30 3.83 3.03

On other hand a PPR batch 2 which underwent only two hours of secondarydrying had no detectable virus after 14 days of incubation at 45° C.

TABLE 4 Thermostability test of PPR Batch 2 Virus titre after storage atvarious Temperatures Day of Incubation 4° C. 25° C. 37° C. 45° C. 0 5.405.40 5.40 5.40 3 5.33 4.80 4.57 3.90 7 5.33 4.77 4.40 2.70 10 5.40 4.773.97 2.50 14 5.27 4.50 3.60 0.00

The dehydration of Rinderpest and PPR viruses using the anhydrobioticprocedure described, produced a glass-like, honeycombed structure ofapproximately 10% residual moisture within one hour. It is hypothesisedthat the observed exotherm after 40 minutes drying, might indicate theglass transition temperature of the trehalose excipient under reducedpressure where the trehalose changes from a liquid and forms ametastable glass (see Robert J. Williams and A. Carl Leopold, The glassystate in corn embryos, Plant Physiol., 1989, 89, 977-981). The productin this state with a residual moisture content of about 10% had amicro-crystalline structure and exhibited dramatic “flash solubility” onrehydration with diluent. Accelerated thermostability tests on theproduct at 5.36% residual moisture caused unacceptable deterioration asevidenced by huge loss of virus titre (Table 4).

The excipient containing half strength Hanks LYE, 1% FCS and 2.5% w/vtrehalose was sufficient to protect both RP and PPR viruses during theone hour, ultra rapid dehydration, when the residual moisture contentwas rapidly reduced to 10% (Tables 1 and 2).

Exposure to 45° C. for 14 days at 5.36% moisture destroyed the virus(Table 4). However, extension of the secondary dehydration for 17 hourshad the expected effect of further reduction of the residual moisture(to less than 1%), thereby conferring increased thermostability (Table3).

The drop in titre of log 1.9 TCIDI50/ml after exposure to 45° C. for 14days, whilst maintaining a minimum titre of Log₁₀3.03 TCID₅₀/ml comparesfavourably with the expected fall in titre found in the currentlyophilised “thermostable” (Thermovax) vaccines. The complete loss ofvirus in PPR batch 2 which underwent only 2 hours of secondary dryingand then similarly exposed to 45° C., highlights the damaging effect ofa high residual moisture content and emphasises the necessity ofextending the secondary drying to ensure a low residual moisturecontent.

Example 3

Peste des Petits Ruminants (PPR) virus was propagated in vero cells inGMEM medium (Sigma No. G6148) containing 10% foetal bovine serum and 10%tryptose phosphate broth (Difco). The cells were propagated in 150 cm²flasks at a concentration of 26×10⁴-28×10⁴ cells per ml adding 60 ml ofcell suspension per flask.

Each 150 cm² flask was inoculated with sufficient PPR seed virus at amultiplicity of infection (MOI) of 10⁻³ virus particles/cell.

On day 4 post inoculation when early cytopathic effects (CPE) is notedthe medium was replaced with Hanks lactalbumin yeast extract containing2% foetal calf serum with 0.1% trehalose dihydrate (in place ofglucose). This was to reduce the solute concentration and minimiseosmotic stress during subsequent dehydration and to remove reducingsugars, such as glucose, which can contribute to the Maillard reactionwhich during dehydration can denature nucleoproteins.

On day 6 post inoculation, the cpe was 80-90%, the virus fluid washarvested and frozen overnight. The frozen fluid was thawed to liberateendogenous virus and stored at −20° C.

The thawed virus fluid was mixed with 16% w/v sterile aqueous trehalosedihydrate (DFS Ltd) in a 1:1 ratio to give a final concentration oftrehalose of 8% in the virus excipient mixture.

One ml volumes of the virus excipient mixture were distributed intosterile 5 ml vaccine vials (1 ml of moisture into each vial) and thevials were partially stoppered with vented dry butyl rubber stoppers(freshly dried at 130° C. for 3 hours). The dehydration was carried outusing an Edwards Super Modulyo freeze dryer.

The freeze dryer was prepared in advance before loading the shelf withthe vials. The condenser was switched on and the condenser temperaturewas allowed to reach the operational limit of 40° C. The shelf heatingwas switched on and the shelf temperature was set to 40° C. Thecondenser door and the drain valve were closed and the vacuum pumpswitched on with full gas ballast. When the shelf temperature reached40° C. the vials were loaded onto the shelf and a temperaturethermocouple was inserted into a vial on each shelf and the chamber doorclosed with the macro and micro air admittance valves fully open.

When the temperature of the contents of the vials reached 35° C. themacro air admittance valves was partially closed to achieve a pressureof 800-900 mbar. During the first 15 minutes the excipient in the vials,which contained dissolved CO₂ from the bicarbonate in the medium, wasgently degassed. During this phase access of trehalose to the viruslipid membrane could occur.

The macro air admittance valve was further closed to reduce the pressuredown to 500 mbar. The temperature of the excipient in the vials wasallowed to fall to approximately 4.0° C., indicating that evaporativecooling was taking place. The micro air admittance valve was carefullyclosed, while monitoring the excipient temperature, to stabilise thetemperature around 4.0° C. This being the most critical phase in theoperation, care was taken to avoid sputtering andlor a too rapidevaporation which would result in the freezing of the product. Sterileair was metered into the chamber to provide a pressure gradient toencourage the flow of water vapour to the condenser. The producttemperature throughout was maintained at 4° C. by careful control of themicro air admittance valve. After maintaining the temperature of theproduct in the range of about 4° C. for 20 to 30 minutes the micro airadmittance valve was slowly closed while taking care to maintain theproduct temperature of about 4° C. and a chamber pressure of 300 mbar.At this stage the product volume was considerably reduced to a syrupyconsistency. The micro air valve was, by this time, fully closed.

After approximately 40 minutes from the beginning of the primary dryingprocedure the product temperature was seen to rise. After 45 minutes thebulk of the water in the excipient had been evaporated and the productexpanded into a microcrystalline honeycomb structure accompanied by asudden exotherm of approximately 15° C. as the trehalose vitrified intoa metastable foamed glass matrix. Following this the temperature of theproduct fell as further moisture in the product evaporated to a give aproduct having a final moisture content of 10%.

With both macro and micro admittance valves closed and vacuum pump gasballast valve closed, drying was continued overnight, at a chamberpressure of 0.1 mbar, during which time the product temperature reached40° C., the temperature of the shelf in the chamber. Drying wascontinued to complete a total drying time of 24 hours in which the shelftemperature in the chamber was raised 1.0° C. per hour for the last 4hours of the drying time culminating in a final temperature (shelf andproduct) of 44° C. While maintaining a chamber pressure of 0.06 mbar thevials were sealed and capped with aluminium seals.

Samples of the dehydrated product produced according to this examplewere stored at 45° C. for 14 days and the virus titre was determined asdescribed above in Example 2. The residual moisture content of theproduct dehydrated according to the above procedure was determined to beapproximately 1.0%. Following storage at 45° C. for 14 days samples ofthe product were found to have lost only a slight degree of viruspotency and were suitable for producing useful vaccine material.

Example 4

The above procedure of Example 3 was followed for the production ofstabilised RP by using RP virus culture instead of the PPR culturedescribed in Example 3. The drying procedure similarly producedstabilised RP with relatively low loss of titre after being stored at45° C. for 14 days.

What is claimed is:
 1. A method of preserving a biologically-activematerial comprising a live virus or mycoplasma which method comprisesthe steps: (i) mixing an aqueous suspension of the biologically-activematerial with a sterile aqueous solution of trehalose to give atrehalose concentration in the mixture in the range of from 2.5 to 8%w/v; (ii) subjecting the mixture to primary drying, for 30 to 60minutes, at a pressure of less than atmospheric pressure and at atemperature initially no greater than 37° C., and which is controllednot to fall to 0° C. or below and which finally is no greater than 40°C. to form a glassy porous matrix comprising glassy trehalose having aresidual moisture content of not greater than 10% and containing, withinthe matrix, desiccated biologically-active material; and (iii)subjecting the glassy porous matrix of step (ii) to secondary drying for10 to 30 hours at a pressure not greater than 0.1 mbar and at atemperature which finally is in the range of from 40 to 45° C. to form atrehalose matrix having a residual moisture content of not greater than2% containing, within the matrix, desiccated biologically-activematerial.
 2. A method according to claim 1, wherein secondary drying instep (iii) is carried out for 20 to 30 hours.
 3. A method according toclaim 2, wherein secondary drying is carried out for 15 to 17 hours at atemperature of about 37° C. and the temperature is, thereafter, raisedgradually over the remaining secondary drying time to a finaltemperature in the range of from 40 to 45° C.
 4. A method according toclaim 1, wherein the primary drying in step (ii) is carried out at apressure of not greater than 800 mbar.
 5. A method according to claim 1,wherein the residual moisture content of the glassy trehalose matrix atthe end of the primary drying in step (ii) is about 10%.
 6. A methodaccording to claim 1, wherein the residual moisture content at the endof the secondary drying in step (iii) is 1.0% or lower.
 7. A methodaccording to claim 1, wherein the live virus is selected from Rinderpestvirus, Peste des Petits Ruminants virus, Measles virus, Mumps virus,Rubella virus, Yellow Fever virus, Polio virus and Newcastle Diseasevirus.
 8. A method according to claim 7, wherein the live virus isRinderpest virus or Peste des Petits Ruminants virus.
 9. A methodaccording to claim 1, wherein the mycoplasma is Contagious BovinePleuropneumonia mycoplasma.